Antigen receptors are assembled from their component gene segments by a series of highly regulated site- specific DNA recombination reactions known as V(D)J recombination. The over-arching goal ofthis research program is to understand in molecular detail the mechanisms underlying the regulation ofthe V(D)J recombinase.Two lymphocyte restricted proteins, RAGl and RAG2 form a complex with one another and recognize pairs ofrecombination signal sequences (RSSs) that flank rearranging gene segments. The RAGs first nick the DNA then introduce dsDNA breaks. Appropriate broken ends are then joined to each other through the action of DNA repair proteins resulting in the formation ofcoding and signal joints. Targeting ofthe recombinase correlates with transcription ofthe unrearranged gene segments and is regulated at the level of RSS accessibility within chromatin structure. Errors in recombinase targeting are associated with chromosomal translocations that result in lymphoid malignancy and defects in V(D)J recombination result in immunodefiency. We propose to 1) test the idea that a gradient of H3K4me3 modified chromatin positioned by the distal germline k promoter determines the efficiency of Igk locus recombination and is important for allelic exclusion; 2) perform a retroviral cDNA library screen for chromatin modifiers that influence recombination; 3) use new high throughput DNA sequencing technologies to extend our studies ofssDNA nicking and RSS end insertion during V(D)J recombination in vivo; 4) examine in detail how a critical transcription factor, E2A, alters chromatin structure to promote recombination in the Igk locus; 5) use gene targeting to delete a CTGF binding site in a DNAse I sensitive region in between the IgHC V and D gene segments in order to test whether it regulates V-to-D rearrangement; and 6) test the h5T)othesis that the interaction between upstream promoters and downstream enhancers is necessary to promote coding joint formation by holding broken coding ends together. V(D)J recombination has been extensively studied using biochemical systems. Our approach is to take what has been learned in vitro and use it to frame and test hypotheses regarding the regulation of recombination in vivo.